eEF2K Assays for Identifying Compounds that Inhibit eEF2K Activity

ABSTRACT

Assays for identifying novel compounds for inhibiting eEF2 kinase and consequence peptides employed therein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/225,875 filed on Jul. 15, 2009, the contents of which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.5R21RR022859-02 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to assays for identifying novel compoundsfor inhibiting eEF2 kinase and consequence peptides employed therein.The present invention is also directed to kits for practicing the scopeof the present invention.

BACKGROUND OF THE INVENTION

The enzyme Elongation Factor 2 Kinase (eEF2-K) belongs to a novel familyof protein kinases, with prototypical member being Dictyostelium myosinheavy chain kinase A (MHCK A), which display no homology to conventionaleukaryotic protein kinases. This protein kinase is highly specific toElongation Factor 2 (eEF2) and is responsible for eEF2 phosphorylation.eEF2 promotes ribosomal translocation, the reaction that results in themovement of the ribosome along mRNA during translation. eEF2 wasidentified among the most prominently phosphorylated proteins in crudetissue and cell lysates. Importantly, it was found that phosphorylationof eEF2 arrests translation, suggesting that this may be a criticalmechanism by which the rate of protein synthesis is regulated (Ryazanov,A. G. (1987). Ca2+/calmodulin-dependent phosphorylation of elongationfactor 2. FEBS Lett 214, 331-334; Ryazanov, A. G., Shestakova, E. A.,and Natapov, P. G. (1988).)

The activity of this kinase is increased in different types of cancersand may be a valid target for anti-cancer treatment. For example, it hasbeen reported that eEF2-K is overexpressed in breast cancer cell linesand tumors with little or no activity observed in normal breast tissue.Moreover, there is evidence indicating that the enzyme is activated inrat glioblastoma (Chang et al, (1995) Calmodulin-dependant proteinkinases in rat glioblastoma, Cell Growth Diff.) The natural productrottlerin has been shown to inhibit growth of glioma cell lines byinhibiting eEF2-K. Parmer et al, (1997) Cell Growth Differ. Vol. 8,327)).

This enzyme was also shown to have increased activity in human brains ofindividuals with AD (Li, X., Alafuzoff, I., Soininen, H., Winblad, B.,and Pei, J. J. (2005) Levels of mTOR and its downstream targets 4E-BP1,eEF2, and eEF2 kinase in relationships with tau in Alzheimer's diseasebrain. FEBS J.272, 4211-4220) although the mechanism and relevance ofthe enzyme for such purposes was not clear. Accordingly, eEF2 kinaseinhibitors could be used to modulate the pathophysiology of a number ofdisease states including brain cancer, breast cancer, ischemic heartdisease, etc.

It was recently discovered that inactivation of a ubiquitous cellularenzyme eEF2 kinase can confer resistance to radiation by suppressingradiation-induced apoptosis. Mucosal damage, such as the damage to theintestine is a major dose-limiting event in radiation therapy andchemotherapy. Aspects of rapid cell turnover, distinctcompartmentalization of damage, and known differentiation pathways ofcrypt cells in the murine and human intestine have been well studied.Treating such conditions have been the subject of an ongoing research.Various modalities, such as antioxidant therapy and inhibition ofserotonin activity at the gastric level, have been suggested andemployed in the art. However, there is still a need to mitigate sideeffects associated with drug and radiation therapy.

Until now all alpha kinase phosphorylation sites in protein substratesidentified fall within the structural class of alpha helices (Drennanand Ryazanov, 2004). It has been unclear whether these kinases haveprimary sequence specificity dictated by residues that surround the siteof phosphorylation, or whether substrate recognition is mediatedprincipally by secondary structure. Accordingly, there is a need in theart to clarify this issue as a means for developing targeted activeingredients.

SUMMARY OF THE INVENTION

The shortcomings of the prior art are now addressed in the presentinvention to provide a screening assay that can ascertain the degree ofinhibition of eEF2 kinase by a test agent. The present inventionprovides for methods of for an inventive assay that for identifyingsuitable compounds for inhibition of eEF2 kinase. The present disclosurealso describes a novel mechanism of action of eEF2 kinase.

Another aspect of the present invention is directed to the consensuspeptide substrate eEF2 kinase for phosphorylation by eEF2K. In at leastanother embodiment of the present invention a method of screening foractivity of modulating agents in eEF2 kinase pathway is developedemploying a consensus sequence for phosphorylation by eEF2K having a Lysor an Arg residue at the +3 position, and a basic residue or Ser or Thrat the +2 position.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “about” will mean up to plus or minus 5% of theparticular term.

As used herein, “consisting essentially of” refers to variations in theamino acid sequence of the substrate that would not change the bindingrate or extent of the substrate to eEF2K by more than about 5 fold.

The instant invention relates to consensus peptides having a suitablesequence for measuring the activity of eEF2K. In general a peptidesubstrates for eEF2K are defined based on sequence relative to a Thrresidue that is the site of phosphorylation (the zero position). ThePositions C-terminal to the phosphorylation site are identified as the“+” positions, and the positions N-terminal to the phosphorylation siteare the “−” positions. For example, the position immediately C-terminalto the site of phosphorylation is the +1 position, and the position tworesidues C-terminal to the Thr is the +2 position.

In at least one aspect of the present invention, consensus sequenceshave been identified containing Lys or Arg (basic) residue at the +3position, either a basic residue or Ser or Thr at the +2 position, to becritical for phosphorylation by eEF2K. Accordingly, at least one aspectof the present invention provides for polypeptide/protein and eEF2K tobe included in a cell or cell lysates, wherein the consensus sequence isdenoted as set forth in SEQ ID Nos: 1 and 2, respectively.

In another aspect of the present invention, the consensus sequencescontain residues Gln and Glu at the −2 position, charged and hydrophilicresidues at the −1 position. In a more preferred embodiment theconsensus sequence contain at least one of the amino acids Ala, Ser,Thr, Val, Ile, Leu, Gln or Glu at the +1 position, and basic residues atthe +4 position. In another embodiment the preferred substrate is 90%identical to SEQ. ID. No. 1 or SEQ. ID. No. 2 and contains a minimum of5 and a maximum of 35 amino acid residues. A preferred peptide substratecontains a minimum of 7 and a maximum of 18 amino acid residues and is91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID No.1 or 2.The most preferred substrate sequence is: Ac-RKKYKFNEDTERRRFL (SEQ ID.No. 1) and Ac-RKKYRIVWKSIFRRFL (SEQ. ID. 2) which provides at least anunexpected 5, 10, 15, 20, 25, 30 to 35, and preferably 30 to 35 foldincrease in activity as compared to known substrates such as MU-H havingRKKFGESEKTKTKEFL (SEQ. ID. No. 3).

In another aspect of the present invention, methods of identifying eEF2Kinhibitors and analogues thereof as potential therapeutic means. In atleast one embodiment, eEF2K inhibitory compounds of the presentinvention may be identified using a high-throughput screening assay,such that the assay discussed herein. Specifically, eEF2K can beproduced in large quantities by E. coli, or using any other suitablemeans known in the art.

At least one embodiment of the present invention is directed toscreening suitable test agents for their activity against eEF2K. Inanother embodiment, suitable modulating compounds are identified byproviding a consequence sequence to interact with eEF2 kinase toascertain the decrease in phosphate transfer by the enzyme into thesubstrate. In yet another embodiment of the present invention, suitablemodulating agents increase or decrease the binding between the substrateand the enzyme as compared to the binding in the absence of the testagent, wherein increased or decreased binding between the at least oneprotein or peptide and eEF2 kinase is indicative of a modulating agenteEF2 kinase pathway.

Phosphorylation of a consensus sequence, such as Ac-RKKYKFNEDTERRRFL(SEQ ID NO: 1), for eEF2K activity can be measured and compared withreduced activity seen in the presence of a test compound. In onenon-limiting embodiment, kinase activity is measured in both control andtest batches based on the depletion of ATP. In another aspect of thepresent invention, active eEF2K utilizes ATP when phosphorlyating theconsensus sequence. Thus, a reduction in ATP signals an active kinase.

In a preferred embodiment, the reduction in ATP signals is visuallydetected and quantified by coupling the reaction with a luciferaseluminescence assay, which is ATP dependent. Thus, active kinase willreduce ATP and, thereby, reduce the luminescence detected. Conversely,inhibition of eEF2K by a test compound prevents depletion of ATP, whichis detected as an increased luminescence.

In at least another embodiment of the present claims screening kits fordetermining the activity of eEF2 kinase inhibitors are contemplatedwherein the kit contains the at least one protein or peptide having aconsensus sequence for phosphorylation by eEF2K comprising a Lys or Argresidue at the +3 position, and a basic residue or Ser or Thr at the +2position with eEF2 kinase, preferably those set forth in SEQ ID NO. 1 or2 or at least those having 95 to 99% identical peptide chain, and aphosphate source, other suitable reagents and direction of use.

Without seeking to limit the possible scope of use of the identifiedcompounds are envisioned to be use as a treatment regimen for targetingthe inhibition of eEF2K phosphorylative activity. Such treatmentregimens may include treatment of Alzheimer's Disease or induction ofLong Term Potentiation of Synaptic Transmissions. Result below alsosuggest that eEF2K can be an important component of the apoptoticpathway, and its inactivation can confer resistance to radiation.Accordingly, selective inhibitors of eEF2K can be used as drugs toprotect tissues from cell death caused by radiation.

Another aspect of the present invention is directed methodologies foridentifying said compound that employs both the consensus peptidesubstrates and a high-trough assay.

EXAMPLES Example 1 Discovery of Consensus Peptide Substrates for eEF2K

To determine whether eEF2K is sequence specific, we made use of anarrayed peptide library approach, (Turk Lab, Beth Israel DeaconessMedical Center Dept. of Pharmacology), that systematically evaluates thepreference of a given kinase for every amino acid at each of ninepositions surrounding the phosphorylation site (Hutti et al., 2004).

The method employed a library of roughly 200 distinct peptide mixtures.Each peptide contains a central fixed phosphorylation site (an equimolarmixture of serine and threonine) flanked on either side by degeneratepositions, and a carboxy-terminal biotin tag. For each of nine positionssurrounding the phosphoacceptor site, twenty-two peptide mixtures aremade in which each of the twenty unmodified proteogenic amino acids aswell as phosphothreonine and phospho-tyrosine are fixed. The entirecollection thus consists of 198 (22×9) peptides. These peptidesubstrates are arrayed in a spatially addressable manner in a 384-wellplate and treated with the kinase of interest and radiolabeled ATP.Control reactions are also run in the absence of peptide to determinethe background from kinase alone. At the end of the incubation time,aliquots of each reaction are spotted simultaneously onto a streptavidinmembrane using a capillary pin-based liquid transfer device. Themembrane is immersed in a quenching solution, washed extensively toremove unincorporated label, dried, and exposed to a phosphor screen.This allows the amount radiolabel incorporated into peptide to bequantified, providing an indication of which peptides are the mostefficient substrates for a particular kinase. This in turn indicateswhich amino acids at a particular position influence phosphorylation bythat kinase.

The inventors of the present invention performed initial runs using thepeptide library with eEF2K. The kinase was able to efficientlyphosphorylate peptides within the library, and displayed significantamino acid preferences at several positions. eEF2K is highly selectivefor basic residues at the +3 position, with a secondary preference forbasic and possibly serine/threonine at the +2 position. The eEF2Kphosphorylation motif bears little resemblance to any of the motifsascribed to conventional protein kinases (Pinna and Ruzzene, 1996). Forexample, no previously characterized kinases have principal selectivityfor basic residues at the +3 position as seen with eEF2K.

Using information from peptide screening, the inventors generated theconsensus peptide substrate called eEF2p, bearing a consensus sequencefor phosphorylation by eEF2K (with the sequence Ac-RKKYKFNEDTERRRFL, SEQID. No. 1). For comparative purposes an additional peptide TRPM7p(Ac-RKKYRIVWKSIFRRFL, SEQ. ID. No. 2) was prepared, bearing a consensussequence for phosphorylation by another α-kinase, TRPM7 kinase.

In reactions performed at a single substrate concentration (100 μM), itwas found that both peptides are efficiently phosphorylated by theirrespective kinases. These new peptides are significantly bettersubstrates for their kinases and show an unexpected 30 to 35 foldincrease in activity as compared to previously identified peptide MH-Uused in the art to assay for eEF2 kinase. These preliminary resultsindicate that the present inventors have been able to generate a highlyefficient eEF2 kinase substrate that will form the basis for ourinvestigations of the kinetic mechanism and mode of substraterecognition for this kinase.

Example 2 Development of High-Throughput Screening Assay

Using the obtained peptide substrate (eEF2p) the present inventors thendeveloped a high-throughput screening assay to identify small moleculeinhibitors of eEF2K. Accordingly a biochemical assay of eEF2K in orderto avoid problems associated with cell toxicity was pursued, as well as,the inhibition of targets other than eEF2K. The latter problem could beacute for a cell-based assay, because signal transduction pathwaysincluding eEF2K are not clearly defined and therefore unknown componentscould be inhibited in the assay. eEF2K expressed in high yield in E.coli is a good source of the enzyme for these studies, because it has ahigh activity and it is also stable (Pavur et al., 2000).

A screen using eEF2K peptide substrate was employed based on thedepletion of ATP (Kupcho et al., 2003; Singh et al., 2004). In the nextstep, a coupled luminescent assay using luciferase, an ATP-dependentenzyme was performed. In this scenario, a decrease in ATP concentrationsor levels over the course of the reaction result in less luciferaseactivity. Kinase inhibition is thereby measured as an increase inluminescence.

Example 3 eEF2K Inhibitor Screening

To identify novel eEF2 kinase inhibitors, the inventors have screened10,000 compounds from ChemBridge using the developed high-throughputmethodology. For high-throughput eEF2k inhibitors screening assay,GST-tagged human eEF2k is expressed and purified from E. coli expressionsystem, and specifically designed short peptides mimicking eEF2 aresynthesized and used as substrates. Master Mix (166.67 mM pH 6.6HEPES-KOH, 666.67 μl α-peptide, 0.2 U/μL Calmodulin, 16.67 mM DTT,333.33 μl CaCl₂, 33.33 mM MgCl₂) was prepared to achieve the optimalkinase activities. To start the kinase assay, A 10 minute incubation of1 uL eEF2k, 100 μM small molecular compounds and 3 uL Master Mix werefirst done at room temperature in 96-well plates. After that, 10 μM ATPwas added to each well to continue another 30 minutes incubation at the30° C. incubator. Finally, Kinase Glo Luminescet Kinase Assay Platformwas used to stop reactions and generate luminescence as readout byquantifying the amount of ATP left in solutions. Because inhibition ofeEF2k activity will block its usage of ATP, the compounds with higherreadouts will be selected as candidates for further experiments.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

1. Consensus peptide substrate eEF2p consisting essentially of asequence for phosphorylation by eEF2K comprising a Lys or Arg residue atthe +3 position, and a basic residue or Ser or Thr at the +2 position.2. The peptide substrate of claim 1, further comprising Gln and Glu atthe −2 position, charged and hydrophilic residues at the −1 position. 3.The peptide substrate of claim 2, further comprising Ala, Ser, Thr, Val,Ile, Leu, Gln or Glu at the +1 position, and at least on basic residuesat the +4 position.
 4. The peptide substrate of claim 3, comprising thesequence: Ac-RKKYKFNEDTERRRFL (SEQ. ID. 1)


5. Consensus peptide substrate TRPM7p comprising a consensus sequencefor phosphorylation by the α-kinase, TRPM7 kinase comprising a Lys orArg residue at the +3 position, and a basic residue or Ser or Thr at the+2 position.
 6. The peptide substrate of claim 5, comprising thesequence: Ac-RKKYRIVWKSIFRRFL (SEQ. ID. 2)


7. A method of screening for activity of modulating agents in eEF2kinase pathway comprising: (i) contacting at least one protein orpeptide having a consensus sequence for phosphorylation by eEF2Kcomprising a Lys or Arg residue at the +3 position, and a basic residueor Ser or Thr at the +2 position with eEF2 kinase, in the presence of aphosphate source and at least one test agent, (ii) determining whetherbinding between the at least one protein or peptide eEF2 kinase isincreased or decreased as compared to the binding in the absence of thetest agent, wherein increased or decreased binding between the at leastone protein or peptide and eEF2 kinase is indicative of a modulatingagent eEF2 kinase pathway.
 8. The method of claim 7, wherein said testagent decreases the transfer of phosphate to the peptide substratethereby decreasing the binding between said protein or peptide and theeEF2 kinase.
 9. The method of claim 7, wherein said test agent blocksthe binding of the phosphate source to the substrate and decreasing thebinding between said protein or peptide and the eEF2 kinase.
 10. Themethod of claim 1, wherein as the concentration of the at least oneagent increases, decreasing binding eEF2 kinase to the at least oneprotein or peptide is indicative of the presence of a competitive kinaseligand.
 11. The method of claim 5, wherein the eEF2 kinase signalingpathway is associated with a disorder associated with over-expression ofeEF2-K activity.
 12. The method of claim 7, further wherein the testagent is present in at least two concentrations, wherein oneconcentration of the test agent does not inhibit the binding to saidprotein or peptide and another concentration of the test agent inhibitsthe binding to the at least said protein or peptide.
 13. The method ofclaim 12, wherein said at least one protein or peptide and eEF2 kinaseare in a cell or cell lysate.
 14. The method of claim 7, wherein thesequence is set forth in SEQ ID NO: 1 and
 2. 15. The method of claim 7,wherein the sequence is set forth in SEQ ID NO:
 1. 16. The method ofclaim 7 further comprising a second step of determining the bindingbetween the at least one protein or peptide eEF2 kinase is increased ordecreased as compared to the binding in the absence of the test agent,wherein increased or decreased binding between the at least one proteinor peptide and eEF2 kinase is indicative of a modulating agent eEF2kinase pathway.
 17. A kit for screening the activity of modulatingagents in eEF2 kinase pathway comprising: at least one protein orpeptide having a consensus sequence for phosphorylation by eEF2Kcomprising a Lys or Arg residue at the +3 position, and a basic residueor Ser or Thr at the +2 position with eEF2 kinase, a phosphate sourceand directions for use.